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Home , Intracellular receptor, Receptor (biochemistry), Transcription factor

MECHANISMS of chronic

• Various chronic effects have uniform biochemical basis INTRACELLULAR Biochemical In vivo effects effects RECEPTORS INTRACELLULAR RECEPTORS (for soluble messengers) function in the nucleus as factors to alter the rate of transcription of particular

¾-activated transcription factors ¾crucial role in signaling ¾activation of different responsive elements (genes)

SINGLE mechanism -> SEVERAL effects Types of signaling in multicellular => understanding to mechanisms may predict effects

1) female reproduction disorders receptor 2) male feminisation activation 3) tumor promotion

4) immunomodulations

5) developmental toxicity

Modes of cell- • Intracellular signal molecules are small, lipid- soluble molecules such as , 1. Direct cell-cell or cell-matrix , hormones, . (made

2. Secreted molecules. from ) • These molecules diffuse through plasma and A. Endocrine signaling. The signaling molecules are hormones secreted by endocrine cells and carried through the circulation system to act on nuclear membranes and interact directly with target cells at distant body sites. the transcription factors they control.

B. . The signaling molecules released by one cell act on neighboring target cells.

C. . Cells respond to signaling molecules that they themselves produce (response of the to foreign antigens, and cancer cells).

1 The intracellular (nuclear) receptor superfamily Steroid hormones, , retinoids and vitamin D

HYDROPHOBIC: - Non-polar molecules - Gases -

Sequence similarities and three functional regions

– N-terminal region of variable length; in some receptors portions of this region act as activation domain

– At the center, DNA binding domain, made of a repeat of C4- finger motif – Near the C-terminal end, hormone binding domain, which may act as an activation or repression domain.

• Steroid hormones are often required to dimerize with a partner to activate transcription • Receptors for vitamin D, retinoic acid and thyroid hormone bind to responsive elements as heterodimers • Second component of the heterdimer is RXR monomer (i.e, RXR-RAR; RXR-VDR) Regulation of transcription activity • Regulatory mechanisms differ for hetero-dimeric and homodimeric receptors • Heterodimeric receptors are exclusively nuclear; without ligand, they repress transcription by binding to their cognate sites in DNA • Homodimeric receptors are mostly cytoplasmic in the absence of ligands • Hormone binding leads to nuclear translocation of receptors • Absence of hormone causes the aggregation of receptor as a complex with inhibitor , such as Hsp90

2 Endocrine System The endocrine system includes all the organs and tissues that produce hormones • Includes endocrine glands, which are specialized to secrete hormones • Also organs, like the liver, that secrete hormones in addition to other functions

A hormone is a chemical that is secreted into extracellular fluid and carried by the blood - can therefore act at a distance from source - only targets with receptor can respond 13

Hypothalamo-pituitary axis

- Regulation of hormone synthesis - – releasing hormone (GnRH) - Pituitary – folicle stimulating (FSH) and luteineising hormone (LH)

Feedback Mechanisms Lipophilic Hormones • For hormone secretion regulated by the negative Lipophilic hormones include the steroid loop: when hormones (derived from cholesterol) and gland X releases the thyroid hormones ( + ) hormone X, this stimulates target -As well as the retinoids, or vitamin A

cells to release Cortisol (Hydrocortisone) Thyroxine

hormone Y. When CH2OH OH C O H C there is an excess of H C 3 II COOH HO 3 OH H3C HO O CH CH hormone Y, gland X H3C 2

II NH2 "senses" this and O inhibits its release of O hormone X. 18

3 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

Blood 1. Hormone passes plasma through plasma Lipophilic Hormones membrane Lipophilic hormones

These hormones circulate in the blood bound Plasma membrane to transport proteins

- Dissociate from carrier at target cells Nucleus

2. Inside target - Pass through the and cell the binds to a receptor 3. Hormone-receptor bind to an intracellular receptor, either in in the cytoplasm complex binds to or nucleus hormone on DNA, the cytoplasm or the nucleus 5. Change in protein regulating gene synthesis is transcription cellular response mRNA - Hormone-receptor complex binds to Protein DNA

hormone response elements in DNA 4. Protein synthesis

- Regulate Hormone response element 19 20

Steroid Hormones They include sex steroids (estrogen, , testosterone) corticosteroids ( and mineralcorticoids)

Thyroid hormone, vitamin D3, and retinoic acid have different structure and function but share the same with the other steroids.

• Steroid hormones and thyroid hormone diffuse easily into their target cells • Once inside, they bind and activate a specific intracellular receptor • The hormone-receptor complex travels to the nucleus and binds a DNA-associated receptor protein • This interaction prompts DNA transcription to produce mRNA • The mRNA is translated into proteins, which bring about a cellular effect

Endocrine disruption

• Interference of xenobiotics with normal function of hormonal system

Possible consequences: Disruption of , reproduction, development, and/or behavior.

• Shift in sex ratio, defective sexual development • Low fecundity/fertility • Hypo-immunity, • Malformations

4 and release of hormones Interaction with hormone system STEROIDOGENESIS e.g. modulation of CYP11A and/or CYP19 activities binding to plasmatic transport proteins

e.g. down-regulation of receptor levels

Synthesis binding to nuclear hormonal receptor (HR)

activation of HR Transport Stimulation ( of associated heat shock proteins, formation of homodimers) e.g. modulation of other nuclear receptors (PPAR/RXR, RXR/TR) Interaction with receptors binding of the activated receptor complex to specific DNA motifs - HREs Suppression

Metabolization chromatin rearrangement and transcription of estrogen-inducible genes

effects at the cellular, tissue, organ, , and/or population level

Mechanisms of Endocrine disrupters in the steroid hormones signalling environment? disruption EDCs... • POPs and their metabolites - Illegitimate activation of hormonal receptor (HR) • steroid hormones and their derivatives from contraception pills - Binding to HR without activation • alkylphenols - Decrease of HR cellular levels • organometallics (butyltins) • pharmaceuticals - FSH/LH signalling disruption • pesticides - Changes in hormone

OH OH OH - ER :

HO HO 17-β- estriol • play a key role in female hormone regulation and signalling

• are responsible for metabolic, behavioural and morphologic changes occurring during stages of reproduction

• are involved in the growth, development and homeostasis of a number of tissues

• control the bone formation, regulation of homeostasis, cardiovascular system and behaviour

• regulate production, transport and concentration of testicular liquid and anabolic activity of androgens in males

5 Estrogen receptor: ESTROGEN RECEPTORS - ER-α &ER-β:

• a member of the nuclear hormone receptor superfamily • a ligand – inducible

• subtype: ER-α (in breast, , , liver, bone and cardiovascular system, adrenals, testis and urogenital tract) ER-β (in kidneys, prostate and gastrointestinal tract) ER-γ (in fish)

A/B D F C E Domains AF-1 AF-2

Dimerization Nuclear localization Binding of Hsp90

Synthesis and metabolism of estrogens Mechanism of action of the estrogen hormones Cholesterol

estradiol CYP11A + (20,22-lyáza) + TARGET CELL meziprodukt y Pregnenolon 17α-hydroxypregnenolon Dehydroepiandrosteron plasmatic transport CY P17 CYP17 protein NUCLEUS (17α-hydrox yláza) (17, 20-lyáza) estradiol Progesteron 17α-hydroxyprogesteron Androstendion Testosteron

CYP 21 2-OH-estradiol ER CYP19 (21α-hydroxyláza) (aromatáza) CYP1A1 activation of ER CYP1B1 11-deoxykortikosteron 11-deoxykortisol Estron Estradiol 4-OH-estradiol 17β-HSD Estradiol β ? CYP11 sulfotrasnferáza (11β-hydroxyláza) Steroidní sulfatáza DNA 16-OH-estradiol

protein Kortikosteron Kortisol Estron sulfát new synthesis mRNA biological proteins 18- hydr oxyláz a response Aldosteron

Environmental estrogens (xenoestrogens, exoestrogens) Exoestrogens - examples

are a diverse group of substances that do not necessarily share Natural products Industrial chemicals any structural resemblance to the prototypical estrogen (17β-estradiol) Bisphenol A naringenin Nonionic surfactants but evoke effects resembling those of estrogen Pthalate esters coumestrol endosulfan • estrogenic substances (estrogen ) zearalenone • ANTI-estrogenic substances

Environmental pollutant DEHP DDT kepone Pharmaceuticals PCBs/OH-PCBs Ethinyl estradiol PAHs and dioxins Diethylstilbestrol gestodene norgestrel

6 Exoestrogens - Relative Potencies to bind to ERα (REPs) Toxicity assessment - in vivo and in vitro methods

Chemical group Substance REP

Estradiol 1 Endogenous hormones Estriol 6,3.10-3 Testosteron 9,6.10-6 Cuomestrol 6,8.10-3 Phytoestrogens Genistein 4,9.10-4 Pesticides o,p´-DDT 1,1.10-6 2,4,6-trichlorbiphenyl-4´-ol 1.10-2 PCBs 2,5-dichlorobiphenyl-4´-ol 6,2.10-3 3,3´,5,5´tetrachlorobiphenyl-4,4´-diol 1,6.10-4 alkylphenoles 4-tert-oktylphenol 3,6.10-6 phthalates butylbenzylphthalate 4.10-6

REP (RElative Potencies) of selected compounds related to 17-β-estradiol derived from reporter yeast assay

In vitro ER- mediated effects In vitro assay luciferase reporter assay Estrogen or xenoestrogen • competitive + ER P P Nuclear Factors • assay P ER ER

endogenous protein expression (or activity) assay ER ER ERE-Luc • Protein of ER: DNA Binding Ligand-Independent Activation mRNA • assay

Luciferase

Light “Estrogenic Effects” ER-Responsive Genes

ER- mediated effects In vivo assay luciferase reporter assay • uterotropic assay

Exposure (6 – 24 h) standards / samples • vaginal cornification assay

96 microwell plate cultivation of transgenic cell lines Cell lysis • standard test procedures for reproductive -> extraction of induced luciferase ER: breast carcinoma MVLN cells and developmental toxicity (e.g. FETAX)

SIMILAR DESIGN FOR OTHER RECEPTORS: • production of estrogen-inducible proteins Luminescence determination Lumino (microplate luminescence reader) AhR (H4IIE.luc cells) (e.g. vittelogenin and zona radiata AR (MDA cells) protein) RAR/RXR (P19 cells)

7 (AR) Androgens

- Role in males similar to the of estrogens in females - development of male sexual characteristics - stimulating protein synthesis, growth of bones - cell differenciation, spermatogenesis - male type of behaviour

Androgens Hypothalamo-pituitary axis

- Folicle stimulating hormone - Stimulates synthesis of - Endogenous ligands – androgen hormones androgen binding proteins

- testosterone OH and spermatogenesis in - dihydrotestosterone (DHT) Sertoli cells (testis) - androstanediol - Luteineizing hormone O - Stimulates testosterone - dehydroepiandrosterone production in Leydig cells - androstenedione Testosterone

Testosterone Dihydrotestosterone

- The most important derivative of testosterone -Formedextratesticulary from testosterone -5α-reductase

- synthetized in testis (Leydig cells) - in lesser extent in adrenals

8 Dihydrotestosterone Mechanism of action

- In several tissues (seminal vesicles, prostate, skin) higher affinity to androgen receptor than Androg ens AR testosterone AR - Daily production 5-10% of testosterone OH Androgen-responsive elements

Ge ne expression O Dihydrotestosterone

Mechanisms of androgen signalling Mechanisms of androgen signalling disruption disruption Binding to AR Alterations of testosterone synthesis - Mostly competitive inhibition – xenobiotics do mostly NOT activate AR-dependent transcription - Inhibition of P450scc needed for side chain cleavage of cholesterol (fungicide ketoconazol) - Few compounds are able to activate AR in absence of androgen hormones x in presence of T/DHT antiandrogenic - Inhibition of 17- α-hydroxylase and other CYPs - – (metabolites of fungicide vinclozoline, some PAHs) needed for testosterone synthesis (ketoconazol)

FSH/LH () signalling disruption Testosterone metabolic - FSH/LH expression - regulation via by - Induction of UDP-glucuronosyltransferase or testosterone monooxygenases CYP1A, 1B involved in androgen catabolism - Suppressing leads to alterations of spermatogenesis - Pesticides endosulfan, mirex, o-p´-DDT

Effects of male exposure to Effects of male exposure to antiandrogens

Exposure during prenatal development: - malformations of the reproductive tract Exposure in prepubertal age: Exposure in adult age: - reduced anogenital distance - delayed - oligospermia - hypospadias (abnormal position of the urethral opening on the penis) - reduced seminal vesicles - azoospermia - vagina development - reduced prostate - libido diminution - undescendent ectopic testes - atrophy of seminal vesicles and prostate gland

9 AR-binding - potencies (Ref: DHT EC50 ~ 0.1 uM) Antiandrogenic compounds

Compound IC50 (µM) Benz[a]anthracene 3.2 Benzo[a]pyrene 3.9 tris-(4-chlorophenyl)-methanol Dimethylbenz[a]anthracene 10.4 Chrysene 10.3 - Ubiquitous contaminant of uncertain origin Dibenzo[a,h]anthracene activation in range 0.1-10µM Bisphenol A 5 - Probable metabolite of DDT-mixture contaminant vinclozolin metabolites 9.7 - Levels in human blood serum cca. 50nM hydroxyflutamide 5 Aroclor typical values 0.25-1.11 - EC50 – cca. 200nM Individual PCBs typical values 64 - 87 tris-(4-chlorophenyl)-methanol 0.2

In vivo antiandrogenicity In vitro assessment antiandrogenicity assessment Hershberger assay Most often employed – prostatic cell lines - castrated rats treated with examined substance Cell proliferation assays – cell lines with androgen- dependent growth; - Endpoint – after 4-7 days – seminal vesicles and - Treatment with tested chemical only (androgenicity) ventral prostate weight or cotreatment with DHT (antiandrogenicity) - mammary carcinoma cell lines - prostatic carcinoma cell lines Measurement of testosterone concentration in serum

In vitro antiandrogenicity In vitro antiandrogenicity assessment assessment GFP - Possibility of nondestructive measurement (fluorescence of Receptor-reporter assays intact cells) - Gene for luciferase or GFP synthesis under X transcriptional control of AR Less sensitive – lack of enzymatic amplification - Human prostatic cell lines - Luciferase: Yeast assays - AR-CALUX (human breast carcinoma T47D) - Mostly β-galactosidase as reporter enzyme - PALM (human prostatic carcinoma PC-3) - Easy cultivation and experimental design X - CHO515 (Chinese hamster ovary CHO) - Cell wall may obstruct transport of chemical into cell=> => false negatives

10 Thyroxine Thyroid hormones

Play crucial roles in stimulating metabolism Calcitonin and influencing development and maturation

Regulation of metabolism Thyroid hormones - increasing oxygen consumption - modulating levels of other hormones (, glucagon, somatotropin, adrenalin) - important in cell differenciation - crucial role in development of CNS, gonads and bones

The Thyroid Gland Effects of thyroid disruption Thyroid hormones bind to nuclear receptors Thyroid hormones - regulate carbohydrate & lipid metabolism - if absent during fetal development or for first year: - adults with hypothyroidism have low - nervous system fails to develop normally production of thyroxine - mental retardation results - reduced metabolism and overweight - In prenatal development - severe damage of CNS (cretenism, delayed eye opening, - adults with hyperthyroidism have high cognition) production (excessive secretion) of - Megalotestis thyroid hormones (thyroxine) - Histological changes in thyroid gland (goitre) - high metabolism and weight loss if T4 concentrations decline before puberty: - trigger metamorphosis in amphibians - normal skeletal development will not continue 63

Thyroid hormones Thyroid hormones Triiodothyronine (T3) Thyroxine (T4) - Enter target cells by transport system Also called tetraiodothyronine Contains 3 iodide ions - Affect most cells in body Contains 4 iodide ions - T4 and small amount of T3 produced in thyroid gland OH OH I I I - Most T3 produced by deiodination I I O O in target tissues (deiodinases) - T4 – prohormone I NH2 I NH2 - 5´-deiodination leads - T4 synthesis - iodination of tyrosin residues on tyreoglobulin to active form, T3 O OH O OH - coupling of two iodotyrosines conducted by

Thyroxine (T4) 3,5,3’-Triiodothyronine (T3)

11 Hypothalamus Pituitary-thyroid axis Enzymes involved in thyroid metabolism OH Anterior „outer“ - Regulation of thyroid synthesis I pituitary - Thyroid peroxidases I - Control the secretion of thyroid TSH I hormones through two - iodination of tyrosyl residues O negative feedback loops Thyroid - coupling of iodinated tyrosyl residues I NH2

- Pituitary TSH (thyroid „inner“ OH T + T - Thyroid deiodinases O 3 4 stimulating hormone) stimulates both I- uptake - D1, D2 - activation of T4 into T3 via deiodination on and iodination of „outer“ ring (formation of T3) tyrosine resides on Tg - D3 - deactivation into rT3 via deiodination on „inner“ ring

Thyroid receptors Thyroid binding proteins - Mechanism of action

Thyroid hormones bind to receptors in: - Regulating free T4 and T3 levels in blood ¾ cytoplasm ¾ surfaces of mitochondria ¾ nucleus - 3 types :

Alike other nuclear receptors -Thyroid-binding prealbunin () (20-25%) - 5 isoforms of TR -Albumin (5-10%) - After activation formation of homo- and heterodimers -Thyroid binding globulin (75%) - Binding to thyroid responsive elements (TRE) - Gene expression

Competitive binding to Competitive binding to TR thyroid binding proteins - Probably less important than binding to TBP - Chemicals that affect thyroid signalling in vivo mostly don´t bind to TR (DDT, PCBs) or bind with much lesser affinity - OH-PCBs, brominated and chlorinated flame than T3 (OH-PCBs – 10000x) retardants, DDT, dieldrin - OH-PCBs – equal affinity to TBP as T4 and T3 Accelerated depletion of TH - More of free T4 in blood => negative feedback to ¾UDP-glucuronosyltransferase – detoxication enzyme TSH release => increased depletion => increased (II.biotransformation phase) weight, histological changes in thyroid gland (after ¾ Induced by PCBs, dioxins ¾ Key enzyme in thyroid catabolism exposure to POPs in mammals, birds, fish) ¾Increased by disruption of TBP binding

12 In vivo assessment In vitro assessment

- Enzyme inhibition assays (thyroid peroxidase, deiodinases) – - TH serum levels – simple, nondestructive x variation within assessment of thyroid metabolism time of day, age, sensitive to other than biochemical stresses

- Thyroid gland weight and folicular cells number - Competitive binding assays with TBP - Developmental toxicity assays - delayed eye opening, abnormalities in brain development and cognition, increased - TH- dependent proliferation assay (pituitary tumor GH3, thyroid testis weight and sperm counts tumors like FRTL-5 cell line) or TSH-dependent proliferation - discharge test (TH synthesis) assay (thyroid tumors) - Hepatic UDP-glucuronosyltransferase activity (marker of - Receptor-reporter gene assays with luciferase (monkey kidney CV- enhanced TH clearance from serum) 1, chinese hamster ovary CHO or insect Sf9 cell lines)

Retinoids

Regulation of development Important for , and homeostasis in tissues of and Retinoids vertebrates and invertebrates differenciation Development of embryonic, Antioxidative epithelial cells (gastrointestinal agent Vitamin A and its derivatives tract, skin, bones) Necessary for vision Affect nervous and immune function Suppressive effects in cancer development

Retinoids RE: Retinol-Ester Sources: from diet (dietary hormones) R: Retinol Retinyl esters – sources

Plant carotenoids CH3 RBP: Retinol Binding CH3 CH3 CH3 CH3 Protein (LMW) β-karoten

CH3 CH3 CH3 CH3 TTR: Transthyrethin CH 3 Bond cleavage (HMW)

Retinol (vitamin A) Retinoic Acid

CH3 CH3 CH3 CH3 CH3 CH3 O

OH OH

CH3 CH3 CH3 CH3

13 Retinoidy RAR Mode of action RAR RXR

RXR - Isoforms of RAR a RXR RARE RXRE - Both have isoforms α, β and γ, each of them several subtypes Exprese - Formation of homo- and heterodimers genů - 48 possible RAR-RXR heterodimers =>sensitive regulation of gene expression - RXR – heterodimers even with other receptors like VDR, TR, PPAR

Retinoic acid

Retinoid binding proteins CRBP – cellular retinol binding protein - 3 basic subtypes - binding of retinol, immediate decrease of retinol - all-trans-, 9-cis- and 13-cis-retinoic acid concentration - All-trans RA binds selectively to RAR CRBAP – cellular retinoic acid binding protein RAL - Retinal - Controlling ratio free retinol/free retinoic - Cis RA bind to both receptor types acid

Disruption of Consequences of retinoid signalling by xenobiotics signalling disruption - Relatively little is known Decreased retinoid levels in organisms - Possible modes of action: - Downregulation of growth factors - Xerophtalmia, night blindness - Metabolization of retinoids by detoxication enzymes - Embryotoxicity, developmental abnormalities X - Disruption of binding retinoids to retinoid binding proteins Increased ATRA concentration – teratogenic effect - Retinoids as antioxidants may be consumed cause of oxidative stress caused by xenobiotics Change may cause severe developmental anomalies - Interference of chemicals (binding to RAR/RXR) (both excess and deficiency)

Disruption of retinoid signalling by xenobiotics Tests to assess retinoid signalling disruption Polluted areas – mostly decrease of retinoid levels in aquatic In vivo birds, mammals and fish - Mostly derived from classical toxicity tests, particularly of Disruption of retinoid transport: PCBs developmental toxicity Effects on retinoid receptors: - Direct measurements of various retinoid forms in living organisms - RAR, RXR binding and/or transactivation – pesticides (laboratory and wildlife) (chlordane, dieldrin, methoprene, tributyltin…) In vitro - Effect on ATRA mediated response – TCDD, PAHs - Mostly epithelial cell lines () - Mouse embryonic cell lines P19 Disruption of retinoid metabolism: pluripotent cells – PCDD/Fs, PAHs, PCBs, pesticides differentiation dependent on circumstances, triggered by ATRA - changes of serum concentrations of retinol and RA - reporter gene assay P19/A15 - Other cell lines – rainbow trout gonads, human salivary gland, - mobilization of hepatic storage forms breast or prostatic carcinomas etc. - in kidney, concentration of all forms elevated

14 AhR (Arylhydrocarbon receptor) ?? Physiological role for AhR → Effects in AhR-deficient mice:

AhR structure Denison et al., Chem. Biol. Interact. 141: 3 • significant growth retardation; • defective development of liver and immune system; • retinoid accumulation in liver; • abnormal kidney and hepatic vascular structures.

• resistant to BaP-induced carcinogenesis and TCDD-induced teratogenesis; • no inducible expression of CYP 1A1 and 2.

Biological responses to TCDD AhR Schmidt & Bradfield, Annu. Rev. Cell Dev. Biol. 12:55

• ligand-activated transcription factor • activation of different responsive elements (genes) • important of toxicity of POPs – primary target of coplanar aromatic substances • regulator of xenobiotic metabolism and activation of promutagens • crossactivation/crosstalk with other receptors • strongest known ligand TCDD

AhR = cytosolic helix-loop-helix/PAS protein AhR domain structure: PAS proteins:

Denison et al., Chem. Biol. Interact. 141: 3

15 AhR activation:

AhR regulated genes:

contain xenobiotic response elements (XRE) or dioxin responsive elements (DRE) in their region:

• phase I enzymes - CYP 1A1, CYP 1A2, CYP 1B1; • phase II enzymes - UDP-glucuronosyltransferase, GST- Ya, NADP(H):oxidoreductase; • other genes - Bax, p27Kip1, Jun B, TGF-β - regulation of cell and apoptosis;

„Non-classical“ AhR ligands

Denison & Nagy, Annu. Rev. Pharmacol. Toxicol. 43:309

Detection of EROD activity: Biomarkers/bioanalytical methods:

• in vivo: liver enlargement, reduction of thymus weight, wasting syndrome, reproductive and developmental disorders • in vivo biomarkers: EROD activity, CYP 1A1 and 1B1 expression; • in vitro: Î EROD in H4IIE rat hepatoma cells; Î CALUX/CAFLUX assays; Î GRAB assay (AhR-DNA binding) Î yeast bioassay; Î immunoassays; Î detection of CYP1A mRNA or protein

16 In vitro assays for nongenotoxic (epigenetic) effects Comparing compounds AhR-mediated effects -> Application in Risk Assessment luciferase reporter assay - H4IIE.luc cells • Quantification of effects (EC ) - relative potencies Ligand (TCDD) 50 1 • Comparison with the effect of reference toxicant (2,3,7,8- + AhR Src ARNT HSP90 TCDD) HSP90 Nuclear • Expression as Equivalency Factors (~ TEFs) P Factors ARNT HSP90 2 AhR TCDD: IC 120 TCDD 50 HSP90 Src Estrogens DRE-Luc 4´-OH-PCBB[a]P 79 4´-OH-PCB 3 PAH: IEC50 “Activated” B[e]P [AhR <-> 100 ER] P Increased Protein Modulation of Gene 80 Induction Equivalency Factor Phosphorylation Expression IEF = IC50 /IEC50 Membrane 60 Proteins P 40 How many times is the compound Cytosolic IEC50 = 2μ M Proteins "weaker" inducer than TCDD ? Light Luciferase 20 IC50 = 10 pM AhR-mediated activity (%TCDD-)

Adapted from Blankenship (1994) 0 1.E-07 1.E-04 1.E-01 1.E+02 CALUX/CAFLUX assays concentrationμ (M)

Toxic equivalency factors (TEF)/TEQ concept: Toxic equivalency factors for PCDDs, PCDFs and PCBs: TEFs provide a simple, single number that is indicative of overall toxicity of a sample containing a mixture of dioxins and dioxin- like compounds. TEFs are consensus values based on REPs across multiple species and/or endpoints. TEFs are based upon a number of endpoints, from chronic in vivo toxicity to in vitro toxicity with the former having the greatest importance in determining overall TEF.

The total of a mixture can be expressed in TCDD TEQ concentration:

Eljarrat & Barceló, Trends Anal. Chem.22: 655

In vitro assays for nongenotoxic effects Cross-talk between estrogen signalling pathways Nuclear Receptors & Signalling Crosstalk and other receptors poorly characterized (toxicity) mechanisms

Nuclear receptors (AhR, ER, RAR/RXR ...) = Transcription factors with • estrogen signalling pathways and numerous cofactors and interactions (crosstalk) other members of superfamily CH3 CH3 H3CCH3 COOH

CH OH 3 RAR ? • estrogen signalling pathways and AhR HO ER AhR • estrogen signalling pathways and receptors for EGF and insuline P P CH3 CH3 hsp90 P H3CCH3 hsp90 RAR COOH ER CH3

P hsp90 hsp90 AhR ER AhR AhR RAR ER RAR

17 In vitro assays for nongenotoxic effects

Modulation of RAR/RXR : retinoic acid signalling

CH3 CH3 H3CCH3 COOH

ATRA – important regulatory molecule CH3 : (embryotoxicity, teratogenicity), other biological events Concentrations of retinoids are known to be modulated by PCBs (? mechanism)

In vitro assay for modulation of ATRA - RAR/RXR effects Luciferase reporter gene assay (embryonic P19/A15) CH3 CH3 H3CCH3 RAR- dependent gene transcription COOH RAR CH3 ? ER

CH3 CH3 AhR H 3CCH3 COOH

RAR CH Luciferase 3

18